A system to enhance the sensitivity of digoxigenin-labelled probe: detection of B19 DNA in serum samples

A highly sensitive dot-blot hybridisation assay for the routine screening o f numerous samples is described. using parvovirus B19 as a model. Digoxigen in-labelled B19 DNA probe was constructed by PCR. hybrids were detected by an anti-digoxigenin monoclonal antibody followed by a second step. using an ti-mouse antibodies conjugated to an alkaline phosphatase-dextran complex ( EnVision(TM). Dako) was carried out. The sensitivity of the assay was evalu ated using both colourimetric and chemiluminescent substrates for the alkal ine phosphatase and was compared with a dot-blot hybridisation assay using the digoxigenin-labelled probe and a standard detection system. With the co lourimetric substrate. the EnVision(TM) system was able to detect 10 fg of B19 DNA. while with the chemiluminescent substrate the sensitivity increase d by up to fg (6 x 10(2) genome copies). This detection system was shown to increase the sensitivity of the assay compared to the standard colourimetr ic visualisation for the digoxigenin-labelled probe. which could detect 0.1 pg. On account of its sensitivity and specificity the dot-blot hybridisati on assay together with the chemiluminescent substrate for the EnVision(TM) detection system was used to analyse 760 serum samples: the same sera were tested for B19 DNA with the standard colourimetric visualisation for the di goxigenin-labelled probe used routinely in the diagnostic laboratory. (C) 2 001 Elsevier Science B.V. All rights reserved.